EP0324603A2 - Festphasen-Enzymimmuntest-Vorrichtung und Verfahren zum Nachweis von Antikörpern - Google Patents

Festphasen-Enzymimmuntest-Vorrichtung und Verfahren zum Nachweis von Antikörpern Download PDF

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Publication number
EP0324603A2
EP0324603A2 EP89300229A EP89300229A EP0324603A2 EP 0324603 A2 EP0324603 A2 EP 0324603A2 EP 89300229 A EP89300229 A EP 89300229A EP 89300229 A EP89300229 A EP 89300229A EP 0324603 A2 EP0324603 A2 EP 0324603A2
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EP
European Patent Office
Prior art keywords
antigen
nitrocellulose membrane
membrane
specific
porous
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Application number
EP89300229A
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English (en)
French (fr)
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EP0324603A3 (de
Inventor
Carole Ann Golden
Albert Jerome Wilson
Melree K. Black
Richard M. Kuts
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GENERAL BIOMETRICS Inc
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GENERAL BIOMETRICS Inc
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Publication of EP0324603A2 publication Critical patent/EP0324603A2/de
Publication of EP0324603A3 publication Critical patent/EP0324603A3/de
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/548—Carbohydrates, e.g. dextran

Definitions

  • the present invention relates to immunochemical systems and processes for determining the presence of antibodies or antigens in a clinical sample and relates most closely to a procedure known as enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • both the cited prior invention and the present invention each relate to protein dot blotting systems classified as an immunochemical technique and more specifically an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • protein spotting This procedure is generally referred to as protein spotting or, as in the present invention, dot-­immunobinding and is distinct from the earlier protein blotting from a gel matrices in that in such spotting procedures a measured aliquot of protein suspension is applied directly to the surface of the membrane and allowed to absorb into the membrane.
  • protein blotting from a gel matrices involves placing the fractionating gel matrices containing the immobilized proteins in direct contact with the nitrocellulose membrane and allowing transfer of proteins from the gel matrices to proceed by capillary action and often require a great deal of time of several hours to several days.
  • the dot-immunobinding technique of the present invention consists of applying a measured amount as a dot of protein solution onto the surface of the nitrocellulose membrane. Thereafter, such membrane is preferably subjected to a blocking reaction where an agent is applied thereto to block protein binding for proteins other than those being tested for. After blocking, the spotted membrane will be used for detection of a specific antibody by subjecting the membrane to antigen-antibody reaction conditions.
  • a discussion of earlier antibody detection procedures is set out in the earlier cited patent application Serial No. 857,643.
  • both the present invention and the above-cited earlier application are directed to protein dot-immunobinding techniques. They each involve a unique utilization of an immobilized nitrocellulose membrane in the form of a dip stick or stick support. They differ from other ELISA methods in that the colored products identifying a positive reaction are insoluble rather than soluble, and each is capable of testing whole blood. Additionally, unique to the present invention, the stick support configuration of the present invention allows for greater ease with which the immunochemical reactions may be executed including the handling of the membrane. With closely controlled heat bonding of the preferred nitrocellulose membrane to a backing, the finished stick support will not include contamination as was possible with the adhesive bonding process as was formerly practiced.
  • the present invention provides a system that, like the earlier invention, allows for very short incubation periods of the stick support in a reagent, as opposed to an earlier standard of thirty (30) minutes. Also unique, the present invention includes a use of reagents that have been tabletized that are to be reconstituted when ready for use and accordingly have a much longer shelf life than do liquid reagents. The present invention also utilizes an improved "stick" holder and vessel structure for use therewith that are unique. And finally, the system of the present invention employs an improved washing step between incubations.
  • the present invention provides a more efficient and reliable blood serum testing system for use in the setting of a doctor's office to test, in a single procedure, for the presence of antibodies to a number of different antigens.
  • Another object of the present invention is to provide, as the test apparatus, a porous membrane that is mounted by heat bonding onto a rigid support and whereto dots of specific antigens are separately immobilized that will, after sequential incubations in different immunochemical reagents, with washes between, individually react in the presence of a specific antibody by changing color, thereby visually indicating the presence of such specific antibody.
  • Another object of the present invention is to provide long life tabletized reagents that are preferably activated by an addition of a light NaCl solution to provide a reconstituted reagent wherein are performed the sequential incubation steps.
  • Still another object of the present invention is to provide a vortex wash apparatus for use in a wash step between incubations.
  • Still another object of the present invention is to provide an improved apparatus and procedure for simultaneously detecting, in a short period of time, the presence of specific antibodies in a clinical specimen in the setting of a medical doctor's office.
  • the present invention is an improved enzyme-linked dot blot immunoassay technique for simultaneously detecting the presence of antibodies to several different antigens from a clinical specimen.
  • the improved apparatus of the invention includes a porous membrane, preferably nitrocellulose, that is immobilized by heat bonding it onto a plastic support. In such heat bonding, the contact pressure and temperature on the nitrocellulose and plastic over time are closely controlled to just induce a flow therebetween, producing a welding of the layers together without causing a change in the physical characteristics of the nitrocellulose. Prior to heat bonding the backing will have had holes formed at intervals therein that serve as windows, exposing the porous nitrocellulose membrane therethrough.
  • an array of purified antigens are dot blotted and dried onto the porous nitrocellulose membrane, each approximately centered in one of the separate windows or compartments.
  • a stick support mounting the immunodotted immobilized membrane is thereby formed to be held on one end for use in dip stick fashion. So arranged, the stick support is to be sequentially immersed in certain reagents with light NaCl washes between for detecting, as color changes on the purified antigen dots, antibodies from clinical samples. To provide this detection, three five (5) minute incubation steps are required. All of which incubation steps are performed at ambient temperature. The sequential incubations are performed in three separate containers that individually receive tabletized reagents.
  • the reagents are each preferably activated by introduction of a NaCl solution thereto.
  • Each vessel to receive, in turn, the nitrocellulose membrane end of the stick support inserted therein, whereafter the stick support membrane end is subjected to a vortex wash.
  • the first container is to contain a sample specimen diluent in tablet form that is activated or reconstituted by mixing a NaCl solution therewith along with a drop of either plasma, serum, or whole blood from a clinical specimen.
  • the stick support is inserted into this diluent and sample for a five (5) minute incubation period during which time certain antibodies as are present in the clinical sample will bind to certain of the antigen spots.
  • the stick support is inserted into a second container.
  • the second container preferably contains, in tabletized form, an alkaline phosphatase conjugated anti-human immunoglobulin G. Prior to stick support insertion this tablet is reconstituted by introduction and mixing of a NaCl solution therewith. During this incubation step enzyme conjugate binds to antibodies as were absorbed from the clinical sample bound during the first step. Following another vortex wash utilizing the vortex tube containing the same or a new NaCl solution the stick support membrane end is immersed in a third and final container that preferably contains chromogenic substrates for the alkaline phosphatase in tabletized form that is reconstituted by mixing a preferred volume and concentration of a NaCl solution.
  • the alkaline phosphatase enzyme converts the soluble chromogenic substrates into colored insoluble products than bind to the porous nitrocellulose membrane at the site of the bound enzyme conjugate.
  • the bound products are thereby each revealed as a colored spot on the nitrocellulose membrane surface.
  • the absence of a colored spot indicates the absence of the particular antibody in the clinical sample.
  • the improved enzyme-linked dot blot assay application of the present invention has been found to consistently, accurately and simultaneously identify the presence of antibodies to a variety of antigens in a single procedure that can be practiced in a short period of time.
  • the test procedure preferably employs a stick support 10 whereon are arranged an array of dot-immunobound purified antigens.
  • the stick support 10 includes, as shown in Fig. 1, a support 11 that is preferably a long rectangular thin wafer of rigid plastic of approximately 40 mil thickness with an array of round or ellipsoid windows 12 formed at spaced intervals.
  • the windows 12 are preferably beveled inwardly at 13 to approximately fifteen degrees from a right angle and are arranged at intervals from a lower end 11.
  • the individual windows are each shown labeled with characters below that window as are appropriate to identify the particular antigen dot fixed in the window above.
  • a porous membrane 14, preferably nitrocellulose, is attached by heat bonding as illustrated in Fig. 1, to support 11 to form the stick support 10.
  • support 11 and membrane 14 are pressed together for a short period of time at a pressure and temperature that are closely controlled to introduce a slight melting of the junction of each of the surfaces. This melting is just sufficient to provide a welding together of the layers without a disruption of the nitrocellulose membrane physical characteristics as could effect the ability of the nitrocellulose to serve as a transfer membrane.
  • the layers are subject to temperature of approximately two hundred twenty degrees Fahrenheit, plus or minus twenty degrees Fahrenheit, and a pressure of approximately thirty (30) pounds per square inch, plus or minus three (3) pounds per square inch for approximately one (1) second plus or minus point two (0.2) seconds, for a nitrocellulose membrane layer and polyvinylchloride support. Should, however, another membrane material or different plastic support be utilized, the preferred heat bonding temperature and pressure would have to be adjusted for the particular material or materials.
  • Characters 15, shown as P, N, W, X, Y, and Z, in Figs. 1 and 2 are preferably formed to the support 11, positioned on line underneath each window 12 for identifying the particular window or dot therein.
  • a roughened surface is preferably provided on a stick support handle end 11b, as shown best in Fig. 2, to provide a surface for receiving writing or the like thereon allowing an operator to mark the particular stick support 10, for clinical sample identification.
  • Such control as is spotted in this window 12 consists of a buffer solution as is used for the solubilization/dilution of all the other samples that are spotted in the lettered windows, or can be a negative control antigen such as a cellular extract of uninfected cells as used for viral antigen production.
  • a top window identified as P, adjacent to stick support end 11b can be allocated for receiving a positive control.
  • the positive control is to verify that all reagents are functioning properly and that the test procedures have been properly executed. Such control will also verify that the three reaction vessels, shown in Fig. 3, are sufficiently reconstituted to the desired volume to successfully perform the test procedure. In practice, this window has been spotted with a solution of purified human immunoglobulin G (lgG).
  • the windows 12, identified at W, X, Y, and Z are preferably spotted with various purified antigens that are each specific for a type of antibody to be tested for.
  • the total number of types of antibodies to be tested for determines the number of windows 12 as are needed.
  • a stick support 10 that incorporates six windows, two for the positive and negative controls with the other four windows spotted with various purified antigens, has been used successfully.
  • nitrocellulose membrane-bound end 11a of the stick support 10 is blocked or capped, as illustrated in Fig. 2 by the centrally holed disks 17 that represent coating of the membrane.
  • Blocking is employed to occupy the nitrocellulose with a non-interfering protein, or other like compound that will prevent the non-specific binding of the immunochemical compounds thereto. This blocking is such as to minimally effect the spots 16, and is practiced to limit the high affinity that most porous membranes, such as nitrocellulose, exhibit to non-specific absorption of a wide range of proteins.
  • the membrane-bound end 11a of the stick support 10 is preferably immersed in a solution containing non-fat dry milk in a Tris-saline solution (5% non-fat dry milk in 10mM Tris, pH 7.4, 0.9% NaCl) for approximately fifteen (15) minutes at ambient temperature.
  • Tris-saline solution 5% non-fat dry milk in 10mM Tris, pH 7.4, 0.9% NaCl
  • Other blocking solutions may be employed to include solutions of albumin, caesin, gelatin, detergents, amino rich compounds, and others.
  • the time and temperature that the membrane is exposed to the blocking agent may be varied depending on the membrane employed and the desired level of acceptable sensitivity/­interference. Further, blocking may be omitted entirety for some applications where a lower level of sensitivity is acceptable.
  • stick support 10 is briefly rinsed in distilled water and is allowed to air dry at ambient temperature.
  • Other rinse solutions may also be employed such as buffered or detergent containing solutions where greater levels of sensitivity for a particular test configuration are desired.
  • Blocked stick supports 10 can be packaged in foil pouches and stored at refrigerator temperature until use, or they may be packaged in other suitable containers or not packaged at all depending on their anticipated use. Packaged or unpackaged stick supports 10 may also be stored at ambient temperature for some applications. Further, while not shown, two such stick supports 10 can be manufactured or arranged together in back to back configuration to double the number of specific antibodies that can be tested for in a single procedure.
  • Fig. 3 illustrates a preferred practice of the process of the present invention as including successive incubation steps of the stick support 10 of Figs. 1 and 2 in containers 1, 2 and 3 with rinses between incubations.
  • a stick support 10 prepared as set out above, is successively incubated at ambient temperature for five (5) minutes in each of three separate immunochemical solutions that are contained, respectively, in containers 1, 2 and 3, the stick support also shown as being rinsed, preferably in a light salt (NaCl) solution, between each incubation.
  • the steps shown in Fig. 3 are preferably performed under ambient conditions other incubation temperatures and times may also be employed to effect a speeding up or slow down of an incubation within the scope of this disclosure.
  • the process of the present invention is preferably performed in small plastic containers, each to contain an immunochemical solution.
  • Each container is to receive the nitrocellulose bound membrane end of the stick support 10 fitted therein.
  • the containers 1, 2 and 3 are preferably empty prior to its receiving a tablet 20 of immunochemical reagents dropped therein, as illustrated in Fig. 5.
  • each tablet is a 100 mg tablet, that is added to the empty dry container and, preferably is reconstituted with 2 ml of 0.1 M NaCl solution.
  • a disposable plastic pipette Prior to the immersion of the stick support membrane end therein, a disposable plastic pipette, not shown, may be used to stir the contents of each vessel to insure the dissolution of the individual tablet to provide adequate mixing of the resuspended reagents, the pipette is moved so as to force the liquid contents of each container up and down several times as needed.
  • the reagent tablets are prepared from the materials described below. In that preparation appropriate absorbed materials are dried and then blended with the other chemical reagents. The blends are then tabletized according to standard tabletizing procedures common to the tabletizing industry to include mixing and compression molding or stamping. The tablets are prepared from bulk quantities of the chemical reagents as listed below which recipes will each produce approximately one one hundred mg tablet of each reagent. Of course, in practice the tablets are preferably compounded in large batches and the weights of materials are accordingly increased proportionally for the number of tablets to be produced. Additionally, a variety of modifications of the reagent recipes are possible within the scope of this disclosure. Such modifications may be employed to enhance tablet disintegration time, stability, cost, and activity of the critical immunochemical reagents. Because of their shelf life, packaging and handling ease and low production cost tabletized immunochemical reagents are more desirable than lyophilized reagents.
  • the preferred constituents of the tablets for each vessel are as follows:
  • the first of the three containers, numbered 1 preferably contains a clinical sample diluent that is provided to dilute the clinical sample provided by a patient to a less concentrated form and will also provide blocking agents to minimize non-specific binding of the clinical sample to the membrane.
  • the preferred reconstituted diluent for vessel 1 is as set out above.
  • other solutions may also be employed as diluent such as, for example, Tris-saline, phosphate buffered saline, detergent containing solutions, and the like, providing such other solution minimizes non-specific interactions of the immunochemical reagents.
  • a drop of either serum, plasma, or whole blood is added to that container 1 and the container constituents are mixed thoroughly.
  • a very small or very large drop of blood or several drops of blood may be added to the sample diluent without affecting test results. (An average drop is approximately 45-50 microliters).
  • coagulation problems may be avoided by employing the diluted blood sample with relative haste such that the sample will have been used and discarded prior to the onset of coagulation.
  • an anti-coagulant may be included as part of the reagent or added to the reconstituted diluent. During this first incubation step, if present, a specific antibody from the clinical sample will bind to the spot of that specific antigen.
  • the above step 1 includes incubating the stick support 10 in the presence of the clinical sample and sample diluent, illustrated as arrow A. After an incubation for approximately a five (5) minute period the stick support is removed from the container 1 and briefly rinsed. The rinse is preferably performed by fitting the stick support in a vortex tube 19, shown in Fig. 3, step 2. To provide for this wash step, shown in Fig. 4A the stick support 10 top end is fitted by sliding it into an appropriate slit cut laterally in an undersurface of a stopper 18 that is preferably formed of neoprene polypropolene, or a like material. The edges of that lateral cut are to flex apart, allowing passage of the stick support end to fit therebetween and then flex back to grip that stick support end.
  • a pier 18a whereagainst one end edge of the stick support end will come to rest. So arranged, the other stick support edge will essentially align with the neoprene stopper circumference.
  • an operator can thereby align and lower the stick support 10 mounted to stopper 18 into a vortex wash tube 19 that is partially filled with a rinse solution, preferably a 0.1 M NaCl solution, or the like.
  • Fig. 4C shows the stopper mounted stick support 10 inserted into the vortex wash tube 19, with the stopper 18 at its outer circumference flexed against to seal over the tube open end.
  • the stick support 10 is positioned, as shown in Fig.
  • the stick support is subjected to a vortex rinse in a 0.1 M NaCl solution between each incubation (performed in vessels 1 and 2).
  • this wash step can also be performed after a last incubation (performed in vessel 3).
  • the stick support 10 is positioned, as shown in Fig. 4C such that the one edge butts against the vortex tube 19 wall, with the stopper 18 holding the stick support in this position and sealing over the tube end.
  • a vortex wash is preferred for all rinse steps and is to provide an acceptable level of sensitivity/­interference of the now activated dots 16, which wash may, but need not, be performed in the same wash solution.
  • the stopper mounted strip support be relatively free of vibration. While vortexing the vortex tube 19 therefore, it is helpful for the operator to hold the tube between the thumb and third finger with the index finger placed firmly on top of the stopper 19. Should the strip support 10 begin to vibrate back and forth during the vortex washing the index finger may be used to apply a slight downward pressure on the stopper so as to urge the slit edges closer together, clamping against the stick support end, to prevent further vibration.
  • the neoprene stopper 19 may be replaced with another like device provided such other device can be used to perform the same function, that of retaining the antigen dotted membrane of the stick support firmly in place during the vortex washing process.
  • the rinse or wash solution may be varied both as to volume and composition. It is, however, important that NaCl be used in the solution though a variety of concentrations are adequate, and a solution of 1 M - 0.1 M NaCl solution is preferred. Such NaCl solution, it has been found, enhances the intensity of the immunochemical reactions thus permitting a more intense visual indication upon completion of the test.
  • the vortex washing procedure is desirable in that it is highly reproducible, rapid, produces an enhanced immunochemical reaction sensitivity, utilizes a minimum volume of washing solution, and has consumer appeal.
  • the stick support 10 is inserted in container 2.
  • This container preferably contains a reconstituted conjugate preferably produced from mixing approximately 2 ml of 0.1 M NaCl solution with the tabletized conjugate.
  • a preferred conjugate is as set out above.
  • conjugates of other specificities it should be understood, are also within the scope of the present invention.
  • Use of an enzyme conjugate essentially provides an enzyme-linked immunosorbent assay (ELISA) based procedures as set out in the above-cited earlier patent application of three of the present inventors, and so will not be further discussed herein.
  • ELISA enzyme-linked immunosorbent assay
  • the incubation of the stick support 10 in vessel 2 is followed by another rinse, that is illustrated as the vortex tube 19 shown in step 3 before arrow C of Fig. 3.
  • this wash is conducted essentially the same as the vortex washing procedure set out hereinabove.
  • the wash solution used in the first wash can be reused, or a fresh wash of NaCl solution may be used.
  • the presence of NaCl in this second wash solution is not critical, it may be omitted without effect and distilled water used in its place. It is, however, desirable to reuse the first wash solution as it minimizes the quantity of reagents to be packaged and simplifies the test procedure.
  • the stick support 10 is installed into the third container that contains enzyme substrates for the enzyme conjugate that will react with the conjugate in such a way as to visually reveal the presence of said conjugate.
  • the preferred enzyme substrate is that shown above and is reconstituted by mixing that enzyme substrate in its tabletized form with approximately 2 ml of 0.1 M NaCl solution. It should, however, be understood that other enzyme substrates may be substituted within the scope of this disclosure so long as such would produce a visible color change to a particular dot 16 on the stick support nitrocellulose membrane.
  • the preferred alkaline phosphatase will interact with the NBT/BCIP substrates.
  • step 3 Shown as the product of step 3, that is a result of this enzymatic interaction, is the production and deposition of insoluble colored precipitates on the nitrocellulose membrane 14 at the site of the bound enzyme conjugate dot 16. This deposition and its tight binding to the membrane produces on the stick support 10 a blue-violet colored spot 16 against a white background of the nitrocellulose membrane 14 within the ellipsoid window 12. This incubation may, but need not, be followed with a brief rinse that is again preferably a vortex wash in NaCl solution or distilled water.
  • Colored spots that have appeared within one or more of the windows after the third incubation indicate the presence of a specific antibody for that specific antigen in the clinical specimen, with the absence of such colored spot indicating that such specific antibody is not present.
  • examination of the positive and negative control windows P and N should show a presence of a colored spot in the preferred positive control window with no detectable spot in the preferred negative control window. Results other than these for the two controls invalidates the test. This is because a color change of the positive control and no color change of the negative control will indicate that all test reagents worked properly at the time of testing and that the proper procedures for executing the test were followed.
  • the present invention and the dot blot system of the prior application for testing for the presence of one or more antibodies has been found in practice to have several advantages over earlier serological procedures. Specifically, that: the test can simultaneously identify the presence of antibodies to several antigens; the test can be performed using a single drop of whole blood; the test does not require highly trained personnel and sophisticated instrumentation; the test can be done very rapidly, in less than twenty (20) minutes; the test preferably includes its own built in positive and negative controls; the test is easily read; and, the tested stick support can be kept as a permanent record.
  • An improved test apparatus and process for its use to simultaneously detect the presence of a variety of specific antibodies in a clinical specimen wherein the process involves an enzyme-linked dot blot immunoassay where an array of specific antigens that are used to detect specific antibodies are separately immobilized as dots (16) on a porous membrane (14) that is preferably porous nitro­cellulose which is itself immobilized by heat bonding on a rigid support (11).
  • the immobilized nitrocellulose membrane is used in the form of a stick support (10).
  • Specific antibodies are detected by sequential incubations of the antigen stick support in three different solutions of immunochemical reagents that are reconstituted with a 0.1 M NaCl solution from tabletized constituents.
  • the stick support (10) is subjected to a vortex wash in a vortex tube (19), containing a light NaCl solution, between incubations and, after the final incubation, the presence of specific antibody is evidenced by the appearance of a blue-violet colour change to a dot of a specific antibody immobilized on the porous nitrocellulose membrane with the absence of colour development indicating the absence of such specific antibody.

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EP19890300229 1988-01-11 1989-01-11 Festphasen-Enzymimmuntest-Vorrichtung und Verfahren zum Nachweis von Antikörpern Withdrawn EP0324603A3 (de)

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EP0427017A2 (de) * 1989-10-31 1991-05-15 S P A SOCIETA' PRODOTTI ANTIBIOTICI S.p.a. Feste Formulierungen für Bioassays, die Avidin oder analoge Substanzen und einen biotinylierten Marker enthalten
EP0478047A1 (de) * 1990-09-17 1992-04-01 Johnson & Johnson Clinical Diagnostics, Inc. Testvorrichtungen
WO1993008474A1 (en) * 1991-10-25 1993-04-29 Cytech Biomedical, Inc. Solid support and assembly for quantitative and qualitative characterization of antigens
FR2699930A1 (fr) * 1992-12-24 1994-07-01 Kabore Paul Réactifs et procédés pour le dosage d'éléments réactifs et autres cofacteurs d'une réaction.
US5472883A (en) * 1992-02-05 1995-12-05 Yamasa Corporation Solid phase reagent and assay method for measuring antibodies specific to antiphospholipid syndrome
WO1997026539A1 (en) * 1996-01-16 1997-07-24 Beckman Instruments, Inc. Analytical biochemistry system with robotically carried bioarray

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Publication number Priority date Publication date Assignee Title
US7297552B2 (en) 2004-04-08 2007-11-20 Sysmex Corporation Instruments for forming an immobilized sample on a porous membrane, and methods for quantifying target substances in immobilized samples
US7749775B2 (en) * 2006-10-03 2010-07-06 Jonathan Scott Maher Immunoassay test device and method of use

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EP0063810A1 (de) * 1981-04-29 1982-11-03 Ciba-Geigy Ag Prüfmittel und Ausrüstung für immunologische Analysen
EP0174247A2 (de) * 1984-08-23 1986-03-12 Bernard Guerin Band zur immunologischen Bestimmung und Verfahren zu seiner Herstellung
EP0301141A1 (de) * 1987-07-30 1989-02-01 Microbiological Research Corporation Testvorrichtung für Festphasen-Enzymimmunoassay und Verfahren zur Bestimmung von Antikörpern

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Publication number Priority date Publication date Assignee Title
EP0063810A1 (de) * 1981-04-29 1982-11-03 Ciba-Geigy Ag Prüfmittel und Ausrüstung für immunologische Analysen
EP0174247A2 (de) * 1984-08-23 1986-03-12 Bernard Guerin Band zur immunologischen Bestimmung und Verfahren zu seiner Herstellung
EP0301141A1 (de) * 1987-07-30 1989-02-01 Microbiological Research Corporation Testvorrichtung für Festphasen-Enzymimmunoassay und Verfahren zur Bestimmung von Antikörpern

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Title
JOURNAL OF IMMUNOLOGICAL METHODS, vol. 62, 1983, pages 325-329, Elsevier Science Publishers B.V.; V. HOREJSI et al.: "Nitrocellulose membrane as an antigen or antibody carrier for screening hybridoma cultures" *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0427017A2 (de) * 1989-10-31 1991-05-15 S P A SOCIETA' PRODOTTI ANTIBIOTICI S.p.a. Feste Formulierungen für Bioassays, die Avidin oder analoge Substanzen und einen biotinylierten Marker enthalten
EP0427017A3 (en) * 1989-10-31 1991-07-31 S P A Societa' Prodotti Antibiotici S.P.A. Solid formulations of complex systems like avidin substances-biotinylated marker, useful for bioassays
EP0478047A1 (de) * 1990-09-17 1992-04-01 Johnson & Johnson Clinical Diagnostics, Inc. Testvorrichtungen
WO1993008474A1 (en) * 1991-10-25 1993-04-29 Cytech Biomedical, Inc. Solid support and assembly for quantitative and qualitative characterization of antigens
US5472883A (en) * 1992-02-05 1995-12-05 Yamasa Corporation Solid phase reagent and assay method for measuring antibodies specific to antiphospholipid syndrome
FR2699930A1 (fr) * 1992-12-24 1994-07-01 Kabore Paul Réactifs et procédés pour le dosage d'éléments réactifs et autres cofacteurs d'une réaction.
WO1997026539A1 (en) * 1996-01-16 1997-07-24 Beckman Instruments, Inc. Analytical biochemistry system with robotically carried bioarray
AU734126B2 (en) * 1996-01-16 2001-06-07 Beckman Instruments, Inc. Analytical biochemistry system with robotically carried bioarray
AU734126C (en) * 1996-01-16 2002-01-03 Beckman Instruments, Inc. Analytical biochemistry system with robotically carried bioarray
US8273304B2 (en) 1996-01-16 2012-09-25 Affymetrix, Inc. Analytical biochemistry system with robotically carried bioarray

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JPH01223352A (ja) 1989-09-06
EP0324603A3 (de) 1990-11-22

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